The present invention relates to methods of killing cells by down-regulating CKI and use of same in prevention and treatment of cancer.
The Wnt pathway is highly conserved throughout evolution, from worms to man, playing crucial roles in embryonic development and diseases. Wnt signaling is strictly regulated by a set of kinases and phosphatases, acting on different components of the cascade and leading to various cell fates during an organism's life.
The main target of the canonical Wnt pathway is cytoplasmic β-catenin, which serves as a transcription co-activator for genes of proliferation, differentiation, migration and survival. The transduction of signal depends on the presence or absence of the Wnt ligand. In resting tissues, in the absence of Wnt ligand, β-catenin is constantly phosphorylated and degraded by a multiprotein complex, and is thus maintained at low levels in cells. In dividing cells, in adult's self-renewing tissues and throughout embryogenesis, secreted Wnt proteins bind to members of the Frizzled receptor family and to the coreceptor LRP5/6 on the cell membrane. Wnt binding activates Dishevelled (Dv1), resulting in dissociation of β-catenin degradation complex and stabilization of β-catenin in the cytoplasm. This enables the translocation of β-catenin into the nucleus and the activation of its target genes (e.g. c-Myc, cyclin D1) through Tcf/Lef-dependent transcription. Deregulation of the canonical Wnt signal leads to various cancers, among which is colorectal carcinoma (CRC), hepatocellular carcinoma (HCC) and melanoma. In such cancers, one or more Wnt component is often mutated, resulting in aberrant accumulation of nuclear β-catenin. This explains the requirement for tight regulation on β-catenin levels in the cell.
The mechanism by which β-catenin is phosphorylated and degraded has been revealed only recently, emphasizing significant players in the Wnt signaling pathway. The β-catenin degradation complex consists of the Adenomatous polyposis coli (APC) tumor suppressor, Axin1 or Axin2 (which are thought to play a scaffold function), and of two Serine/Threonine kinases: Casein kinase I (CKI) and Glycogen synthase kinase-3 (GSK3), which phosphorylate β-catenin on four N-terminal Ser/Thr residues. This event marks β-catenin for ubiquitination by the SCFPβ-TrCP E3 ubiquitin ligase and subsequent proteasomal degradation. It has been shown lately that the first phosphorylation event is mediated by CKI, which phosphorylates Ser45 of β-catenin. This creates a priming site for GSK3, which subsequently phosphorylates Thr41, Ser37 and Ser33. The last two residues, when phosphorylated, serve as a docking site for the E3 ligase βTrCP, which marks β-catenin for degradation.
CKI's involvement was proven to be both necessary and sufficient for driving the cascade leading to β-catenin down-regulation. This is in agreement with studies on Wnt components' homologues in Drosophila and therefore assigns CKI as a Wnt antagonist. On the other hand, developmental studies in Xenopus and C. elegans implicated CKI as a Wnt effector, showing that CKI promotes secondary body axis and embryonic polarity (Wnt effects). Supporting that is the observation that CKI phosphorylates and activates Dv1, another Wnt effector, thereby increasing β-catenin levels.
CKI is a well-conserved family of Ser/Thr kinases found in every organism tested, from yeast to man. In mammals, the CKI family is composed of seven genes (α, β, γ1, γ2, γ3, δ, ε) encoding 11 alternatively spliced isoforms. Members of the CKI family share a conserved catalytic domain and ATP-binding site, which exclusively differentiate them from other kinase families. CKI is a ubiquitous enzyme found in all cells, occupies different sub-cellular localizations and is involved in various cellular processes besides Wnt signaling.
Mutations in the canonical Wnt pathway abrogate its tight regulation resulting in nuclear accumulation of β-catenin, and the execution of an aberrant Wnt transcription program. These mutations occur in approximately 90% of colorectal cancers, as well as in other cancer types, such as hepatocellular carcinomas (HCC), gastric cancers and melanomas. Activating mutations in β-catenin itself have been reported in approximately 10% of colorectal cancers and up to 40% of HCC. Inactivating mutations in the Wnt pathway can occur in Axin1/2 genes and in the APC gene. Axin1 and Axin2 mutations have been found in HCC and colorectal cancer (CRC) respectively, though to a much lesser extent than APC mutations. The APC tumor suppressor gene is a primary target for somatic inactivating mutations in 85% of sporadic CRC's whereas in other types of cancer, APC mutations are rare. Thus the APC mutation, which was initially identified in the inherited cancer syndrome Familial Adenomatous Polyposis (FAP) is the major cause of sporadic CRC and is almost exclusive to this disease, i.e. APC is a colon-specific tumor suppressor gene.
The APC protein is a key regulator of the Wnt pathway. APC tumor suppressor has been shown to participate in several cellular processes including cell cycle regulation, apoptosis, cell adhesion, cell migration, signal transduction, microtubule assembly and chromosomal segregation. However, despite the fact that each of these roles are potentially linked to cancer, it appears that the tumor suppressing function of APC resides primarily in its capacity to properly regulate β-catenin. This effect takes place in two major posttranslational levels, enhancing β-catenin degradation and exporting it from the nucleus. In the absence of functional APC, β-catenin is stabilized and accumulates in the nucleus where it associates with members of the TCF/LEF family transcriptional activators, thus modulating transcription of Wnt target genes. Recent evidence also implicates APC in a nuclear role, suppressing β-catenin-mediated transcription by forming a repression complex on the DNA, thus giving it a third aspect of Wnt regulation.
Consistent with its tumor suppressing role, bi-allelic disruption of the APC gene occurs in both FAP and sporadic CRC. Inactivation of both APC alleles can be detected in most intestinal tumors at early stages of tumor development and the vast majority of APC mutations result in a truncated protein that lack Axin1/2 binding motifs and a varying number of the 20 amino acid repeats that are associated with β-catenin down-regulation.
Stöter et al [Oncogene (2005) 24, 7964-7975], teaches treatment of chiriocarcinomas with an inhibitor of CKI delta.
Yang, W S et al., Genome Biol. 2008; 9(6):R92. Epub 2008 Jun. 2 teaches treatment of cancer with inhibitors of CKI epsilon.
Behrend et al., [Oncogene, 9 Nov. 2000, Volume 19, Number 47, Pages 5303-5313] using specific inhibitors to CKI delta and epsilon teach that both these proteins are essential for an ordered mitotic progression.
U.S. Patent Application No. 20050171005 teaches treating colorectal cancer by providing compositions that up-regulate CKI.
U.S. Patent Application No. 20090005335 teaches treating cancer by providing compositions which down-regulate B-catenin.